10:30 Uhr
P 131:
Abgesagt - Altered promoter methylation of the miR-183/96/182 cluster in human liver is associated with overexpression of miR-182-5p in type 2 diabetes
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Autor:innen:
J. Wagner (Hamburg, DE)
S. Wolter (Hamburg, DE)
O. Mann (Hamburg, DE)
H. Lehnert (Salzburg, AT)
H. Kirchner (Lübeck, DE)
Hypothesis
Type 2 diabetes (T2D) is a multifactorial disease with a genetic and epigenetic component in its pathogenesis. Especially microRNAs are altered in metabolic diseases and regulate key metabolic genes. Therefore, we wanted to analyse the regulatory mechanism of hepatic microRNA expression in T2D.
Methods
We analysed the DNA methylome of liver samples from 28 diabetic and 46 non-diabetic obese subjects collected during bariatric surgery by whole genome bisulfite sequencing (WGBS). Subsequent analysis focussed on CpG sites within genes encoding microRNAs and their putative promoters (2500 bp upstream). Identified differentially methylated CpGs (DMCs) were validated by bisulfite pyrosequencing. Methylation was correlated with microRNA expression quantified by qPCR analysis.
Results
We identified 9,438 hepatic DMCs associated to 1675 microRNAs between obese subjects with and without T2D. Of these, 367 positions were significantly (q < 0.05) hypermethylated and 71 positions were significantly hypomethylated in diabetic liver. Moreover, we identified 24 DMCs within the miR-183/96/182 cluster. Interestingly, altered hepatic miR-182-5p expression was previously associated to clinical parameters of T2D. Pyrosequencing validated that one DMC adjacent to multiple transcription factor binding sites within the promoter of MIR182 was significantly hypermethylated (5.6%, p=0.0099) in T2D and methylation correlated positively with hepatic miR-182-5p expression (p=0.0033, r=0.48).
Conclusion
By performing WGBS for the first time in liver of obese subjects we identified 438 DMCs within genes for microRNAs and their respective promoters. Moreover, one novel DMC within the promoter of MIR182 was hypermethylated and could be a potential cause of hepatic overexpression of miR-182-5p in T2D.
10:37 Uhr
P 132:
One region on chromosome 17 is associated with plasma and pancreatic insulin content in an obese mouse model
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Autor:innen:
M. Delpero (Berlin, DE)
D. Arends (Berlin, DE)
M. Sprechert (Berlin, DE)
G. Brockmann (Berlin, DE)
D. Hesse (Berlin, DE)
Background and aim: The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and related pathologies such as insulin resistance and fatty liver. The aim of this study was to identify genetic variants associated with plasma and pancreatic insulin content in two sub-strains of the BFMI (BFMI861-S1 and BFMI861-S2). These lines are genetically close related but differ in several metabolic phenotypes (e.g. blood glucose concentration, plasma insulin, and body weight).
Methods: An advanced intercross line (AIL) was generated from the cross BFMI861-S1 x BFMI861-S2. In generation 10, plasma insulin and pancreatic insulin content were collected in 397 male mice at 25 weeks under a high fat, high carbohydrate diet. To perform QTL-analysis with the collected phenotypes, genotyping was performed using GigaMUGA SNP chip and KASP assay. Candidate gene prioritization was performed using whole genome sequencing data, gene expression, and proteomics data of the parental lines.
Results: QTL mapping identified one QTL on chromosome 17 (7,725,897 – 26,054,796) associated with plasma and pancreatic insulin content. Candidate gene prioritization identified Acat2 as the most likely candidate gene in this region. This gene carries one deleterious missense variant in the N-terminal domain and shows downregulation at both mRNA and protein level in the gonadal adipose tissue of the BFMI861-S1 line.
Conclusion: QTL mapping and candidate gene prioritization discovered a locus on chromosome 17 involved in the regulation of insulin metabolism. The results confirmed that our BFMI861-S1 line with its unique genetic background is a powerful population to detect metabolic QTL.
10:44 Uhr
P 133:
Leptin expression is dynamically regulated by methylation of three distinct enhancer elements
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Autor:innen:
N. Taege (Lübeck, DE)
S. Schriever (Munich, DE)
C. Geißler (Lübeck, DE)
H. Kirchner (Lübeck, DE)
Hypothesis
The development of leptin-reducing strategies demands the precise understanding of the surprisingly poorly studied regulation of leptin gene expression in response to nutritional status. To address this regulation, we investigated if the DNA methylation of distinct enhancer elements of leptin, as epigenetic answer to nutritional factors, influences leptin gene expression and if this methylation is dynamic.
Methods
First, we screened distal enhancer elements of leptin by pyrosequencing and analyzed leptin gene expression by quantitative real-time PCR in adipose tissues of diet-induced-obese (DIO) mice compared to lean mice for 12 weeks. Second, to investigate the dynamics of leptin gene expression, we analyzed the methylation status of epididymal adipose tissue of mice undergoing a weight cycling program by switching diets from high-fat-diet (HFD) to chow and back to HFD.
Results
We found a substantial change in the DNA methylation of three distinct enhancer elements of leptin depending on the duration of HFD exposure in fat depot specifically. Fat mass, circulating leptin levels and leptin gene expression in the epididymal adipose tissue correlated positively with the enhancer element methylation. The changes in methylation pattern were reversed when DIO mice lost weight by switching them back to chow after HFD exposure. Furthermore, the methylation pattern was re-induced when mice re-gained weight demonstrating the methylation dynamics.
Conclusion
Collectively, this study suggest that DNA methylation of enhancer elements of leptin could regulate leptin gene expression according to nutritional status and body weight. This leptin gene regulation could be exploited to future leptin-reducing weight loss strategies.
10:51 Uhr
P 134:
Finding key-regulator microRNAs in non-alcoholic fatty liver disease progression
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Autor:innen:
J. Britsemmer (Lübeck, DE)
C. Krause (Lübeck, DE)
S. Schriever (Munich , DE)
H. Kirchner (Lübeck, DE)
Aim: With the increasing pandemic of non-alcoholic fatty liver disease (NAFLD), affecting approximately 25% of the world’s population, new treatments and diagnostics are needed. Of great interest are microRNAs (miRNAs) since they promote NAFLD progression by targeting genes involved in hepatic metabolism and are drug targets. miRNAs belong to the epigenetic mechanism of post-transcriptional gene regulation that leads to mRNA degradation or translational repression after binding.
Methods and Results: We hypothesized that a subset of master-regulator miRNAs drive fatty liver progression into non-alcoholic steatohepatitis (NASH). Inhibiting these master-regulators or reversing them through interventions should stop NAFLD progression. Therefore, we combined multiomics approaches of different high-fat diet and five different intervention mouse models. The intervention models contained surgical, diet or treatment intervention. We found 15 differentially expressed target genes that negatively correlated with 122 miRNAs between mice fed a control or high-fat diet. The intervention models showed a partially reversible effect on a subset of micro RNAs and target gene expression. Network analysis revealed two central miRNAs controlling several genes associated with liver health and metabolism, mmu-miR-182-5p and mmu-miR-16-5p. The former also correlates positively with Hba1c, fasting glucose, NAS -and serum triglycerides in liver of obese-diabetic and obese-non diabetic humans.
Discussion: We showed induction and reversal of two hepatic miRNAs by HFD-feeding and weight loss intervention that were associated with genes involved in hepatic steatosis in mice and humans. These findings are crucial for a better understanding of NAFLD development and the identification of novel therapeutic approaches to prevent it.
10:58 Uhr
P 135:
THE FOOD CHAIN PLUS (FOCUS) COHORT: A COHORT FROM NORTHERN GERMANY
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Autor:innen:
C. Geisler (Kiel, DE)
K. Schlicht (Kiel, DE)
C. Knappe (Kiel, DE)
N. Rohmann (Kiel, DE)
K. Hartmann (Kiel, DE)
K. Türk (Kiel, DE)
U. Settgast (Kiel, DE)
D. Schulte (Kiel, DE)
T. Demetrowitsch (Kiel, DE)
J. Jensen-Kroll (Kiel, DE)
A. Pisarevskaja (Kiel, DE)
F. Brix (Kiel, DE)
B. Gruber (Kiel, DE)
G. Rimbach (Kiel, DE)
F. Döring (Kiel, DE)
P. Rosenstiel (Kiel, DE)
A. Franke (Kiel, DE)
S. Schreiber (Kiel, DE)
C. Henning (Kiel, DE)
W. Lieb (Kiel, DE)
U. Nöthlings (Bonn, DE)
K. Schwarz (Kiel, DE)
M. Laudes (Kiel, DE)
Background and aims: Within the last decades the scientific interest increased in the pathology of metabolic-inflammation or metainflammation as predictors of common metabolic diseases. The Food Chain Plus (FoCus) cohort was launched in 2011 for population-based research specifically addressing metabolic-inflammation or metainflammation.
Methods: Participants underwent a comprehensive protocol including anthropometrics, medical history, sociodemographic data, metabolic and inflammatory biomarkers, eating behaviour, sensory perception and social network analysis as well as detailed multiomic and genotype analyses. Data analyses included the data of diseases, clinical biochemistry, nutrition, lifestyle, metabolomics and microbiome composition during baseline and 5-year follow-up.
Results: Data of 1,796 participants (63.1% females and 36.9% males) were available for detailed baseline analyses. The overall prevalences of diabetes mellitus type 2 (T2DM), hypertension and dyslipidaemia were 14.1%, 42.3% and 29.7%. In respect to nicotine abuse, 53.3% of participants with T2DM were former smokers but there was no difference in smoking status between non-diabetic and T2DM participants. Most of T2DM participants were married (69.7%) and pensioners (51.1%). More than 40.0% of T2DM participants showed a reduced quality of life. After 5-year follow-up 4.4% of the participants developed a new diagnosed diabetes and 7.9% improved their diabetes status.
Conclusion: The presented data showed a few results from the cohort. In the near future, more predictors for the identification of metabolic-inflammation/metainflammation will be investigated especially in the development of T2DM through multi-omics statistics regarding different research questions cross-sectional and longitudinal. These analyses will extend the research in obesity and diabetes to the nutrition-gut microbiome-host-metabolism axis.
11:05 Uhr
P 136:
Natural mutations leading to down-regulation of Bbs7 in the Berlin Fat Mouse
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Autor:innen:
K. Mohebian (Berlin, DE)
F. Krause (Berlin, DE)
D. Arends (Berlin, DE)
D. Hesse (Berlin, DE)
R. Kühn (Berlin, DE)
G. Brockmann (Berlin, DE)
Background and Aims: The Berlin Fat Mouse Inbred (BFMI) line carries natural mutations that can help understanding genetic mechanisms causing obesity. The jObes1 locus on chromosome 3 in BFMI explains ~40% of the variance of body weight. The expression of the candidate gene Bbs7 is reduced in brain, adipose tissue and liver. The goal was to identify potential regulatory DNA variants.
Material and Methods: To investigate the genetic effect on expression, we compared the effect of 16 DNA sequence variants between BFMI and C57BL/6N (B6N) in the promoter region and a 1578 bp deletion in intron 8 of Bbs7. Various sequences of the Bbs7 promoter of BFMI and B6N were cloned into dual-luciferase reporter plasmids. Plasmids were transfected into HEK cells in which the luciferase expression served as readout.
To evaluate the role of the deletion in intron 8, CRISPR/Cas9 mice were generated and crossed with mice carrying BFMI or B6N alleles. Complementation tests were performed on the offspring.
Results: All plasmids containing the SNP rs29947545 in the 5‘ UTR of the Bbs7 promoter significantly reduced the expression of Bbs7 in HEK cells (0.35 ≤ Fold Change ≤ 0.51, p≤0.042).
Complementation tests showed that CRISPR/Cas9 modified B6N mice partially complement (13.1 - 15.1 %) the jObes1 allele which was accompanied by a trend towards a reduced Bbs7 expression in these mice.
Conclusion: Both, a 5’ UTR-SNP and a deletion in intron 8 of Bbs7, contribute to the phenotype of the BFMI mice most likely by reducing Bbs7 expression.